pH-Sensitive Gold Nanorods for Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) Delivery and DNA-Binding Studies

A facile experimental protocol for the synthesis of poly(ethylene glycol)-modified (PEGylated) gold nanorods (AuNRs@PEG) is presented as well as an effective drug loading procedure using the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP). The interaction of AuNRs@PEG and drug-loaded AuNRs (AuNRs@PEG@NAP) with calf-thymus DNA was studied at a diverse temperature revealing different interaction modes; AuNRs@PEG may interact via groove-binding and AuNRs@PEG@NAP may intercalate to DNA-bases. The cleavage activity of the gold nanoparticles for supercoiled circular pBR322 plasmid DNA was studied by gel electrophoresis while their affinity for human and bovine serum albumins was also evaluated. Drug-release studies revealed a pH-sensitive behavior with a release up to a maximum of 24% and 33% NAP within the first 180 min at pH = 4.2 and 6.8, respectively. The cytotoxicity of AuNRs@PEG and AuNRs@PEG@NAP was evaluated against MCF-7 and MDA-MB-231 breast cancer cell lines. The development of AuNRs as an efficient non-steroidal anti-inflammatory drugs (NSAIDs) delivery system for chemotherapy is still in its infancy. The present work can shed light and inspire other research groups to work in this direction.


Introduction
Designing nano-platforms where properly functionalized inorganic nanoparticles (NPs) with specific capping ligands and/or bioactive agents are used to enhance biocompatibility, multifunctionality, and explicit targeting characteristics can provide a plethora of new opportunities for a wide range of applications [1][2][3][4][5]. For the case of gold NPs and nanorods (NRs), it has been found that surface functionalization with biomolecules, polymers, fluorophores, thiol-based molecules, and amino acids greatly enhance their properties concerning photothermal therapy [6][7][8][9], cancer theranostics [6], drug/gene delivery [10,11] as well as sensing/imaging purposes [10,11]. For the synthesis of Au NPs/NRs, the seedgrowth technique is widely used while other techniques such as chemical vapor deposition, microwave, electrochemical, and pyrolysis, to name a few, are also presented [10,11]. Concerning the surface functionalization of the Au NPs/NRs, the experimental procedures used involve: (a) effective coating with polymers; (b) immobilization of capping ligands The effective PEGylation of the AuNRs was further supported by the red shift (12 nm) of the l-SPR band of the absorption spectrum of the AuNRs@PEG dispersions [24] as well as the decrease of the ζ-potential from high positive values of 36.7 mV (CTAB-covered AuNR s) to almost close to zero values (~0.6 mV) for AuNRs@PEG [24].
Another confirmation of the effective PEGylation of the AuNR's surface is based on FT-IR spectroscopy. The IR spectra of the AuNRs@PEG@NAP are shown in Figure 2 where three distinctive areas (highlighted with yellow) present bands related to the PEG polymer. More explicitly: (i) the stretching and vibration band of the C-O-C ether is located at 1108 cm −1 (violet region in Figure 2B) and (ii) the out-on-plane vibration band of CH is shown at 956 cm −1 (purple region in Figure 2B) and at 2890 cm −1 ( Figure 2C). The effective ligand exchange between CTAB and mPEG-SH during the PEGylation procedure is also confirmed by the extinction of the two characteristic bands at 2918 and 2848 cm −1 belonging to the asymmetric bands of CH2/CH3 groups of CTAB ( Figure 2C) [25]. Furthermore, the effective loading of NAP in AuNRs@PEG@NAP is also confirmed in the IR spectrum by the characteristic stretching vibration of the carboxylic group at 1724 cm −1 as well as the breathing vibrations of the aromatic rings resolved in the region highlighted with green in Figure 2B. It should be pointed out that using thiol-based PEG derivatives, the ligand exchange reaction is greatly improved leading to the successful elimination of the CTAB and highly efficient PEGylation. The effective PEGylation of the AuNRs was further supported by the red shift (12 nm) of the l-SPR band of the absorption spectrum of the AuNRs@PEG dispersions [24] as well as the decrease of the ζ-potential from high positive values of 36.7 mV (CTAB-covered AuNR s) to almost close to zero values (~0.6 mV) for AuNRs@PEG [24] .
Another confirmation of the effective PEGylation of the AuNR's surface is based on FT-IR spectroscopy. The IR spectra of the AuNRs@PEG@NAP are shown in Figure 2 where three distinctive areas (highlighted with yellow) present bands related to the PEG polymer. More explicitly: (i) the stretching and vibration band of the C-O-C ether is located at 1108 cm −1 (violet region in Figure 2B) and (ii) the out-on-plane vibration band of CH is shown at 956 cm −1 (purple region in Figure 2B) and at 2890 cm −1 ( Figure 2C). The effective ligand exchange between CTAB and mPEG-SH during the PEGylation procedure is also confirmed by the extinction of the two characteristic bands at 2918 and 2848 cm −1 belonging to the asymmetric bands of CH 2 /CH 3 groups of CTAB ( Figure 2C) [25]. Furthermore, the effective loading of NAP in AuNRs@PEG@NAP is also confirmed in the IR spectrum by the characteristic stretching vibration of the carboxylic group at 1724 cm −1 as well as the breathing vibrations of the aromatic rings resolved in the region highlighted with green in Figure 2B. It should be pointed out that using thiol-based PEG derivatives, the ligand exchange reaction is greatly improved leading to the successful elimination of the CTAB and highly efficient PEGylation.

Drug Release Studies
The emission spectra of a solution of free NAP (red line) and the suspension of AuNRs@PEG@NAP (green line) are presented in Figure S2 while no emission was detected for AuNRs@PEG. A comparison between the spectra of the solution of free NAP (λ max,em = 357 nm) and AuNRs@PEG@NAP (λ max,em = 359 nm) reveals a significant broadening and a slight red shift in λ max,em for the latter one. Both of these features can be attributed to changes in the chemical environment surrounding the drug molecule due to its location on the surface of AuNRs@PEG@NAP suggesting a successful loading of the drug. It was possible to calculate the percentage of the drug loading from the calibration curve of the emission intensity of naproxen at 357 nm and it was found to be 112.5 mg/g ( Figure S3). The excitation and emission spectra of an aqueous suspension of AuNRs@PEG@NAP are presented in Figure 3. Due to the fluorescence properties of NAP, the successive loading of the drug on the pegylated surfaces of gold NRs was confirmed by using fluorescence presenting a maximum excitation and emission at 233 nm and 359 nm, respectively, with a large stoke shift of 123 nm [26,27]

Drug Release Studies
The emission spectra of a solution of free NAP (red line) and the suspension of AuNRs@PEG@NAP (green line) are presented in Figure S2 while no emission was detected for AuNRs@PEG. A comparison between the spectra of the solution of free NAP (λmax,em = 357 nm) and AuNRs@PEG@NAP (λmax,em = 359 nm) reveals a significant broadening and a slight red shift in λmax,em for the latter one. Both of these features can be attributed to changes in the chemical environment surrounding the drug molecule due to its location on the surface of AuNRs@PEG@NAP suggesting a successful loading of the In order to check whether targeted drug delivery is pH-sensitive, two representative pH values (4.2, 6.8) were chosen having in mind that the acidic conditions (pH = 4.2) are typical of inflamed tissues. At pH 4.2, the release profile indicates a release up to a maximum of 24% NAP within the first 180 min, while a higher percentage of NAP release (33%) was observed at pH = 6.8 for the same time value (Figure 3). The pronounced burst effect experienced in the first 3 h is possibly related to the desorption of a percentage of the drug close to the surface and its immediate release upon contact with the release medium since PEG is water soluble [28,29]. After the first 3 h, an extremely slow release rate was monitored for both pH values directly related to a change of the release mechanism to interlayer diffusion of protected NAP molecules among the stacking of PEG polymers [30][31][32].
In order to check whether targeted drug delivery is pH-sensitive, two r pH values (4.2, 6.8) were chosen having in mind that the acidic conditions typical of inflamed tissues. At pH 4.2, the release profile indicates a re maximum of 24% NAP within the first 180 min, while a higher percentage o (33%) was observed at pH = 6.8 for the same time value (Figure 3). The pron effect experienced in the first 3 h is possibly related to the desorption of a the drug close to the surface and its immediate release upon contact wi medium since PEG is water soluble [28,29]. After the first 3 h, an extremely rate was monitored for both pH values directly related to a change o mechanism to interlayer diffusion of protected NAP molecules among the sta polymers [30][31][32].
Since this is the first example of an AuNR nano-platform loaded with N there are no direct comparisons with other AuNR drug carriers co loading/release mechanisms. Nevertheless, the release profiles are sim PEGylated ferrite NPs, polymeric capsules and polypeptide vesicles [30][31][32] For the following biomacromolecule-binding studies, the aqueo dispersions of AuNRs@PEG@NAP were prepared in pH = 4.0 and the measu performed in freshly prepared samples and in a time duration of 1-2 h to e loaded NAP on the surface of the gold is not released.

Interaction of AuNRs with CT DNA
DNA is usually a potential biomolecular target for diverse drugs such antiviral, and anticancer agents [33]. The investigation of the interaction Since this is the first example of an AuNR nano-platform loaded with NSAID drugs, there are no direct comparisons with other AuNR drug carriers concerning the loading/release mechanisms. Nevertheless, the release profiles are similar to other PEGylated ferrite NPs, polymeric capsules and polypeptide vesicles [30][31][32].
For the following biomacromolecule-binding studies, the aqueous colloidal dispersions of AuNRs@PEG@NAP were prepared in pH = 4.0 and the measurements were performed in freshly prepared samples and in a time duration of 1-2 h to ensure that the loaded NAP on the surface of the gold is not released.

Interaction of AuNRs with CT DNA
DNA is usually a potential biomolecular target for diverse drugs such antibacterial, antiviral, and anticancer agents [33]. The investigation of the interaction of potentially bioactive compounds with DNA is often employed either complimentary to cytotoxic studies or in order to obtain an insight of possible mechanisms or applications. In general, compounds may interact with DNA in three fashions, i.e., via covalent binding or noncovalent interactions or cleavage of the DNA helix [34]. The interaction of AuNRs@PEG and AuNRs@PEG@NAP with CT DNA was investigated directly by UV-vis spectroscopy indirectly by evaluating their ability to displace EB from the EB-DNA adduct.
UV-vis spectroscopic titrations were employed initially to assess the interaction between CT DNA and AuNRs@PEG and AuNRs@PEG@NAP, in order to gain information on the mechanism and the intensity of this interaction. During such UV-vis spectroscopic titration investigations, the UV-vis spectra of AuNRs@PEG and AuNRs@PEG@NAP were recorded in the presence of increasing amounts of CT DNA. More specifically, in the UVvis spectrum of AuNRs@PEG, the band observed at 525 nm ( Figure 4A) exhibited in the presence of CT a slight hypochromism accompanied by a slight red shift (Table 1). In the UV-vis spectrum of AuNRs@PEG@NAP, two basic bands were observed ( Figure 4B); band I at 530 nm which may be attributed to the presence of a Au-nanorod since it was also found for AuNRs@PEG and band II at 319 nm which may be assigned to the presence of naproxen, as also found in reported naproxen complexes [35][36][37][38][39]. In the presence of CT DNA, these two bands showed a slight hypochromism up to 10% followed by a slight bathochromism (Table 1). Such features may indicate the interaction of AuNRs@PEG and AuNRs@PEG@NAP with CT DNA, although the possible interaction mode may not be concluded safely.
presence of CT a slight hypochromism accompanied by a slight red shift (Table 1). I UV-vis spectrum of AuNRs@PEG@NAP, two basic bands were observed ( Figure 4B); I at 530 nm which may be attributed to the presence of a Au-nanorod since it was found for AuNRs@PEG and band II at 319 nm which may be assigned to the presen naproxen, as also found in reported naproxen complexes [35][36][37][38][39]. In the presence o DNA, these two bands showed a slight hypochromism up to 10% followed by a s bathochromism (Table 1). Such features may indicate the interaction of AuNRs@PEG AuNRs@PEG@NAP with CT DNA, although the possible interaction mode may n concluded safely.  [38,39] 325 (+22, +2) 2.67 (±0.22) × 1 a "+" denotes hyperchromism, "-" denotes hypochromism. b "+" denotes red shift, "-" denote shift.
The binding constants of AuNRs@PEG and AuNRs@PEG@NAP with CT DNA were calculated with the Wolfe-Shimer equation (Equation (S1)) [40] and corresponding plots [DNA]/(εA − εf) versus [DNA] ( Figure S4). At the temperature °C, AuNRs@PEG@NAP presents lower Kb than AuNRs@PEG but higher Kb (almost times) than free naproxen. This may suggest that the incorporation of naproxen i AuNRs@PEG may lead to a tighter interaction with CT DNA than the free NA comparison of the DNA-binding constant of AuNRs@PEG@NAP with the reported m naproxen complexes may reveal that AuNRs@PEG@NAP is a slightly tighter DNA-bi than the Co(II) [37], Cu(II) [38], and most Mn(II) [35,36] complexes of naproxen, similar to Sn(IV) [41] and Ag(I) [42] complexes, while the polyethyleneim functionalized carbon nanotube hosting naproxen [43], the water-soluble silica hy spin-crossover nanoparticle loaded with naproxen [44], and the reported Ni(II)-napr complexes present higher Kb values [39] than AuNRs@PEG@NAP.
The binding constants of AuNRs@PEG and AuNRs@PEG@NAP with CT DNA (K b ) were calculated with the Wolfe-Shimer equation (Equation (S1)) [40] and the correspond- Figure S4). At the temperature of 18 • C, AuNRs@PEG@NAP presents lower K b than AuNRs@PEG but higher K b (almost two times) than free naproxen. This may suggest that the incorporation of naproxen in the AuNRs@PEG may lead to a tighter interaction with CT DNA than the free NAP. A comparison of the DNA-binding constant of AuNRs@PEG@NAP with the reported metal-naproxen complexes may reveal that AuNRs@PEG@NAP is a slightly tighter DNA-binder than the Co(II) [37], Cu(II) [38], and most Mn(II) [35,36] complexes of naproxen, and similar to Sn(IV) [41] and Ag(I) [42] complexes, while the polyethyleneimine-functionalized carbon nanotube hosting naproxen [43], the water-soluble silica hybrid spin-crossover nanoparticle loaded with naproxen [44], and the reported Ni(II)-naproxen complexes present higher K b values [39] than AuNRs@PEG@NAP.
In order to obtain deeper information regarding the possible interaction forces developed between AuNRs@PEG and AuNRs@PEG@NAP with CT DNA, the UV-vis spectroscopic titration studies in the presence of CT DNA were performed for three different temperatures (291 K, 300 K, and 310 K) and the corresponding K b values were also determined ( Table 2). It may be noted that the increase of temperature results in a lower K b value for AuNRs@PEG and a significantly increased K b value for AuNRs@PEG@NAP. Such a trend may suggest different DNA interaction modes for AuNRs@PEG and AuNRs@PEG@NAP. Table 2. Thermodynamic parameters of AuNRs@PEG and AuNRs@PEG@NAP for the interaction with CT DNA at different temperatures (291 K, 300 K, and 310 K). Among the interaction forces developed between a bioactive compound and a biomolecule, the most common ones are hydrophobic forces, electrostatic interactions, van der Waals interactions, and hydrogen bonds [45,46]. The enthalpy change (∆H) and the entropy change (∆S) may be calculated from the Van 't Hoff equation (Equation (S2)) and the plots of ln(K b ) versus (1/T), where -∆H/R is the slope of the fitting line and ∆S/R is the intercept (R is the universal gas constant) ( Figure 5). In addition, ∆G may be obtained from the Gibbs-Helmholtz equation (Equation (S3)).
R PEER REVIEW 8 of 18 EB is a typical DNA intercalator with a known DNA-binding constant (Kb = 1.23 (±0.07) × 10 5 M -1 ) [52]. An indication of its DNA intercalation is the appearance of a strong fluorescence emission band at 592 nm, upon excitation of its solution at 540 nm [53]. A DNA intercalator may displace EB from the EB-DNA adduct resulting in a quenching of the EB-DNA fluorescence emission band. In the present case, the EB-DNA adduct was prepared by the 1 h pre-treatment of a solution containing 20 µM EB and 26 µM CT DNA. The addition of AuNRs@PEG and AuNRs@PEG@NAP in increasing amounts results in a quenching of the EB-DNA band (Figures 6 and S5) which was more intense in the presence of AuNRs@PEG@NAP (up to 46.6% of the initial EB-DNA fluorescence) ( Table  3). This quenching is obviously a result from the displacement of EB from the EB-DNA due to the competition with AuNRs@PEG@NAP for the DNA intercalation sites. Three different combinations of the enthalpy change (∆H) and the entropy change (∆S) have been reported in the literature which are related to the development of different types of interaction between the compound and the biomacromolecule: (a) in the case of ∆H > 0 and ∆S > 0, hydrophobic forces are developed, (b) the combination ∆H < 0 and ∆S < 0 may exist in the case of van der Waals interactions, and hydrogen bonds and (c) electrostatic interactions may lead to ∆H < 0 and ∆S > 0 [46,47]. From the plots ln(K b ) versus (1/T) for AuNRs@PEG and AuNRs@PEG@NAP ( Figure 5), the corresponding ∆H and ∆S values were determined (Table 2). For AuNRs@PEG, both values of ∆H and ∆S are negative revealing the development of van der Waals interactions and hydrogen bonds between AuNRs@PEG and CT DNA which may subsequently indicate the existence of external interactions (groove-binding) with DNA [48][49][50]. In the case of AuNRs@PEG@NAP, both values of ∆H and ∆S are positive suggesting the existence of hydrophobic forces between AuNRs@PEG@NAP and CT DNA, stabilized by π-π stacking interactions which may be explained by the existence of an intercalation [48]. The negative ∆G values for both AuNRs@PEG and AuNRs@PEG@NAP may show that their interaction with CT DNA is spontaneous [46,48,50,51].
EB is a typical DNA intercalator with a known DNA-binding constant (K b = 1.23 (±0.07) × 10 5 M −1 ) [52]. An indication of its DNA intercalation is the appearance of a strong fluorescence emission band at 592 nm, upon excitation of its solution at 540 nm [53]. A DNA intercalator may displace EB from the EB-DNA adduct resulting in a quenching of the EB-DNA fluorescence emission band. In the present case, the EB-DNA adduct was prepared by the 1 h pre-treatment of a solution containing 20 µM EB and 26 µM CT DNA. The addition of AuNRs@PEG and AuNRs@PEG@NAP in increasing amounts results in a quenching of the EB-DNA band (Figures 6 and S5) which was more intense in the presence of AuNRs@PEG@NAP (up to 46.6% of the initial EB-DNA fluorescence) ( Table 3). This quenching is obviously a result from the displacement of EB from the EB-DNA due to the competition with AuNRs@PEG@NAP for the DNA intercalation sites. EB is a typical DNA intercalator with a known DNA-binding constant (Kb = 1.23 (±0.07) × 10 5 M -1 ) [52]. An indication of its DNA intercalation is the appearance of a strong fluorescence emission band at 592 nm, upon excitation of its solution at 540 nm [53]. A DNA intercalator may displace EB from the EB-DNA adduct resulting in a quenching of the EB-DNA fluorescence emission band. In the present case, the EB-DNA adduct was prepared by the 1 h pre-treatment of a solution containing 20 µM EB and 26 µM CT DNA. The addition of AuNRs@PEG and AuNRs@PEG@NAP in increasing amounts results in a quenching of the EB-DNA band (Figures 6 and S5) which was more intense in the presence of AuNRs@PEG@NAP (up to 46.6% of the initial EB-DNA fluorescence) ( Table  3). This quenching is obviously a result from the displacement of EB from the EB-DNA due to the competition with AuNRs@PEG@NAP for the DNA intercalation sites.     The Stern-Volmer plots ( Figure S6) show that the quenching of the EB-DNA fluorescence emission is in good agreement (R 2~0 .99) with the linear Stern-Volmer equation (Equation (S4)), which suggests that the quenching may be assigned to the EB-displacing ability of AuNRs@PEG and especially AuNRs@PEG@NAP. The K SV constants (Table 3) of AuNRs@PEG and AuNRs@PEG@NAP were calculated by the Stern-Volmer equation and the Stern-Volmer plots ( Figure S6). The EB-DNA system has a fluorescence lifetime (τ o ) of 23 ns [54] which is applied for the determination of the corresponding EB-DNA quenching constants (k q ) with Equation (S5) [53]. The k q values of the AuNRs@PEG and AuNRs@PEG@NAP (Table 3) may suggest the existence of a static quenching mechanism for the EB-DNA fluorescence [55]. A direct comparison of the constants of AuNRs@PEG@NAP with those of the reported metal-naproxen complexes may not be achieved due to their expression in the molar scale. Given the molecular mass of NAP (230.2 g/mol), the corresponding constants could be expressed in mg/mL and are much higher than those of AuNRs@PEG@NAP. In addition, the constant of AuNRs@PEG@NAP could be considered comparable with those of a previously reported carbon nanotube and spin-crossover nanoparticle loaded with naproxen [43,44].

Interaction of the AuNRs with pBR322 Plasmid DNA
As a next step to the studies with CT DNA, the cleavage of DNA was investigated in order to evaluate their potential nuclease-like ability. The efficiency of the cleavage of the pBR322 plasmid DNA (pDNA) with AuNRs@PEG and AuNRs@PEG@NAP was visualized and measured by the gel electrophoresis technique. The supercoiled pDNA in an agarose gel during electrophoresis is shown as Form I (Figure 7, Lane 1). In general, a potential cleaving activity by the AuNRs@PEG and AuNRs@PEG@NAP may be revealed as singlestranded nicks (ss) in the supercoiled DNA leading to the formation of the relaxed circular DNA (Form II) and/or double-stranded nicks (ds) forming linear DNA (Form III) and are calculated with Equations (S6) and (S7). All experiments were carried out in duplicate.

Interaction of the AuNRs with Albumins
Serum albumin (SA) is one of the most significant proteins in plasma since it contributes to the transportation of metal ions, drugs, and small molecules through the bloodstream towards their biological targets [53]. Within this context, it is necessary to investigate the affinity of bioactive compounds for albumins; such binding may differentiate the properties of the bioactive compound or provide novel transportation pathways or novel mechanisms of action [56]. For this reason, the interaction of AuNRs@PEG and AuNRs@PEG@NAP with HSA and its homologue BSA was studied by fluorescence emission quenching experiments ( Figure S7 and Figure 8, respectively). When excitation at 295 nm was applied to the initial solutions of HSA and BSA, these solutions exhibited an intense fluorescence emission band at 337-339 nm which is attributed to tryptophan residues, i.e., tryptophan-214 in HSA, and tryptophan-134 and -212 in BSA [53]. The addition of AuNRs@PEG and AuNRs@PEG@NAP at increasing amounts into the albumins solutions resulted in the quenching of the respective band AuNRs@PEG and AuNRs@PEG@NAP were mixed with pDNA (Tris buffer solution, 25 µM, pH = 6.8). After incubation of the components, the pDNA was analyzed by gel electrophoresis on 1% agarose stained with EB. As shown in Figure 7, both AuNRs@PEG and AuNRs@PEG@NAP may induce single-stranded nicks (Form II) up to 48% (Figure 7, Lane 3, for AuNRs@PEG@NAP). In total, the AuNRs presented a rather moderate cleavage activity at a rather high concentration (0.25 mg/mL) with AuNRs@PEG@NAP being marginally more active (48%) than AuNRs@PEG (44%).

Interaction of the AuNRs with Albumins
Serum albumin (SA) is one of the most significant proteins in plasma since it contributes to the transportation of metal ions, drugs, and small molecules through the bloodstream towards their biological targets [53]. Within this context, it is necessary to investigate the affinity of bioactive compounds for albumins; such binding may differentiate the properties of the bioactive compound or provide novel transportation pathways or novel mechanisms of action [56]. For this reason, the interaction of AuNRs@PEG and AuNRs@PEG@NAP with HSA and its homologue BSA was studied by fluorescence emission quenching experiments ( Figure S7 and Figure 8, respectively). When excitation at 295 nm was applied to the initial solutions of HSA and BSA, these solutions exhibited an intense fluorescence emission band at 337-339 nm which is attributed to tryptophan residues, i.e., tryptophan-214 in HSA, and tryptophan-134 and -212 in BSA [53]. The addition of AuNRs@PEG and AuNRs@PEG@NAP at increasing amounts into the albumins solutions resulted in the quenching of the respective band which was much more intense in the presence of AuNRs@PEG@NAP (up to 65.5% of the initial fluorescence of HSA, Figure 9 and Table 4). The inner-filter effect was also checked (Equation (S8)) [57] and it did not affect the measurements.         The quenching constants (k q ) of the interaction of AuNRs@PEG and AuNRs@PEG @NAP with the SAs were determined with the Stern-Volmer quenching equation (Equations (S4) and (S5) [53,58]) where the fluorescence lifetime of tryptophan in SA is taken as τ o = 10 −8 s [58] and from the corresponding Stern-Volmer plots ( Figures S8 and S9). The obtained k q values (Table 4) may indicate the existence of a static quenching mechanism, verifying thus the interaction of the complexes with SAs.
The binding constants (K) of AuNRs@PEG and AuNRs@PEG@NAP with the SAs were determined from the Scatchard equation (Equation (S9)) [53] and the corresponding Scatchard plots (Figures S10 and S11). AuNRs@PEG@NAP presents a significantly higher (two to three times) affinity for both albumins than AuNRs@PEG ( Table 4). The affinity of AuNRs@PEG for albumins is comparable with other gold nanoparticles [59,60] and gold nanoclusters [61]. A comparison of the constants of AuNRs@PEG@NAP with those of the reported metal-naproxen complexes is not possible due to their different concentration expression (the values of the complexes are expressed in the molar scale). Taken into consideration the molecular mass of NAP (230.2), its corresponding constants may also be expressed in mg/mL (Table 4) and, especially the value of K HSA , are much higher than those of AuNRs@PEG@NAP. The binding of AuNRs@PEG@NAP to albumins is reversible and relatively tight when compared to previously reported carriers of naproxen [43,44].

Evaluation of Cytotoxicity
In order to evaluate the biological effect of AuNRs@PEG and AuNRs@PEG@NAP, cytotoxicity studies were performed on two breast cancer cell lines: the low metastatic MCF-7 cells, and the more aggressive MDA-MB-231 cells. More specifically, AuNRs@PEG and AuNRs@PEG@NAP were used at a concentration of 1, 50, and 200 µg/mL and the cells' viability was subsequently determined.
Based on the results, the 1 µg/mL concentration of both AuNRs@PEG and AuNRs@ PEG@NAP showed a slight, non-significant decrease of~5% in cell viability in the MCF-7 cells. Similarly, AuNRs@PEG had the same effect in MDA-MB-231, whereas AuNRs@PEG@ NAP exhibited a higher but not statistical decrease of~13% in cell survival ( Figure 10). Notably, the 50 µg/mL concentration showed a marked decrease in cell survival for AuNRs@PEG and AuNRs@PEG@NAP. At the higher concentration of 200 µg/mL, a significant decrease in cell viability for both AuNRs@PEG and AuNRs@PEG@NAP was also noted. The different cytotoxicity values between these two concentrations may be due to a different inhibitory mechanism or a stimulatory effect in cell growth at higher concentrations. Moreover, at 200 µg/mL the decrease in cell survival was more prominent on the aggressive MDA-MB-231 cells, where AuNRs@PEG decreased cell survival by ca 60% and AuNRs@PEG@NAP by ca 75%. In contrast, both AuNRs@PEG and AuNRs@PEG@NAP on the MCF-7 cells showed a decrease of ca 40%. Overall, both AuNRs@PEG and AuNRs@PEG@NAP exerted a similar effect on cell survival, with higher concentrations resulting in significant effects.
AuNRs@PEG@NAP was also noted. The different cytotoxicity values between these two concentrations may be due to a different inhibitory mechanism or a stimulatory effect in cell growth at higher concentrations. Moreover, at 200 µg/mL the decrease in cell survival was more prominent on the aggressive MDA-MB-231 cells, where AuNRs@PEG decreased cell survival by ca 60% and AuNRs@PEG@NAP by ca 75%. In contrast, both AuNRs@PEG and AuNRs@PEG@NAP on the MCF-7 cells showed a decrease of ca 40%. Overall, both AuNRs@PEG and AuNRs@PEG@NAP exerted a similar effect on cell survival, with higher concentrations resulting in significant effects.
The TEM study was performed utilizing a FEI CM20 TEM operating at 200 kV. TEM specimens were prepared by drop-casting a 3 µL droplet of AuNRs nanoparticles suspension on a carbon-coated Cu TEM grid. The size of the particles were determined by "manual counting" using the ImageJ (v. 1.54d) software (https://imagej.net, accessed on 4 February 2023).
For the interaction of AuNRs@PEG and AuNRs@PEG@NAP with biomacromolecules, different buffer solutions were prepared: (i) buffer solution with pH = 7 (bspH7) containing 150 mM NaCl and 15 mM trisodium citrate where the pH value of 7.0 was adjusted by HCl (aq) , (ii) Tris buffer (25 µM, pH 6.8) and (iii) buffer solution with pH = 4 (bspH4) containing 100 mM CH 3 COOH and 25 mM CH 3 COONa where the pH value of 7.0 was adjusted by HCl (aq) .
The DNA stock solution was prepared by dilution of CT DNA to a buffer solution of pH 7.0 (bspH7) followed by stirring at 4 • C and it was kept at 4 • C for no longer than two weeks. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A 260 /A 280 ) in the range of 1.85-1.90, indicating that the DNA was sufficiently free of protein contamination [62]. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M −1 cm −1 [63]. UV-visible (UV-vis) spectra were recorded on a Hitachi U-2001 dual-beam spectrophotometer. The fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer.

Preparation of AuNRs@PEG and AuNRsPEG@NAP
The synthetic protocol of the seed solution used for the AuNRs growth is the following: 5 mL of HAuCl 4 (0.5 mM) mixed with 5 mL CTAB (0.2 M), while an aqueous solution (1 mL) of 0.01 M NaBH 4 was injected to the previous solution under vigorous stirring. The color of the solution change from yellow to brown-yellow and the stirring was terminated after 2 min. The seed solution was kept at RT for 30 min before use.
To prepare the growth solution, 7.0 g CTAB and 1.234 g NaOL were dissolved in a 1 L flask containing 250 mL of warm water (~50 • C). The solution was left to cool down and then an aqueous solution of AgNO 3 (24 mL, 4 mM) was added and the mixture was kept without stirring for 15 min. Subsequently, 250 mL of HAuCl 4 (1 mM) was added and the solution was stirred for about 1.5 h in order to become colorless. After that, 3 mL HCl (12.1 M) were used to adjust the pH value close to 1.6. Fifteen minutes later, 1.25 mL ascorbic acid (0.064 M) was added under vigorous stirring. Finally, 400 µL of seed solution was added in the resulted mixture and it was left undisturbed at 30 • C overnight in order to form the AuNRs. In order remove the excess of CTAB, the AuNRs were centrifuged at 10,000 rpm (12,298× g) (three times) and the supernatant was discarded while each time the pellet at the bottom of the falcon was redispersed in 15 mL H 2 O.
An amount of 30 mg of mPEG-SH was added in 10 mL of the previously prepared AuNRs solution (O.D. = 2.9, [Au] = 260 µg/mL), which was kept at 29 • C to avoid CTAB crystallization and the resulted solution was stirred for 24 h. The final product was isolated with centrifugation at 5900 rpm (4281× g) for 20 min followed by the removal of the supernatant (unreacted mPEG-SH) and redispersed in 10 mL H 2 O.
For loading the AuNRs@PEG with NSAID NAP, a clear colorless solution of 4 mg of NAP in 2 mL of chloroform was added in the previously prepared solution (10 mL

Study of the Interaction of the AuNRs with Biomacromolecules
Lyophilized AuNRs@PEG and AuNRs@PEG@NAP were initially dissolved in a buffer solution of pH = 4 (100 mM CH 3 COOH and 25 mM CH 3 COONa) at a concentration of 0.25 mg/mL. CT DNA and the albumins were dissolved in a buffer solution of pH = 7 (150 mM NaCl and 15 mM trisodium citrate).
The interaction of AuNRs@PEG and AuNRs@PEG@NAP with CT DNA was examined thoroughly by UV-vis spectroscopy for three different temperatures (291 K, 300 K, and 310 K), and via competitive studies with EB by fluorescence emission spectroscopy. The ability of AuNRs@PEG and AuNRs@PEG@NAP to cleave pDNA was studied by gel electrophoresis. The albumin (BSA or HSA) binding was studied through tryptophan fluorescence quenching experiments. All the specific protocols and relevant equations involved in the in vitro study of the interaction of the AuNRs@PEG and AuNRs@PEG@NAP with biomacromolecules are presented in the Supporting Information file (Sections S1-S3).

Drug-Release Protocol
The emission characterization of AuNRs@PEG@NAP was recorded on a Cary Eclipse emission spectrophotometer. The experimental protocol used for monitoring the drug release behavior was the following: 1 mg of AuNRs@PEG@NAP was dispersed in 5 mL of two buffered solutions (pH = 4.2, 6.8) under stirring at room temperature, achieving final concentration of 0.2 mg/mL. At predetermined time intervals, the solution was transferred in a cuvette and the emission intensity of NAP at 357 nm was recorded. Cumulative release (%) was given in Equation (2): where W t and W drug denote the weights of drug released from the hybrid material at time t, and the total amount of loaded drug, respectively. From the calibration curve of the emission intensity of NAP at 354 nm ( Figure S3), the percentage of the drug loading can also be calculated as: 1 mg of the loaded material was dispersed in 5 mL of buffered solution giving 0.2 mg/mL final concentration. From the maximum emission intensity (184 a.u.) of NAP and the calibration curve, the quantity of naproxen in this dispersion is calculated at 0.022 mg/mL. As this value corresponds to 0.2 mg of the studied material, the loading is calculated at 112.5 mg/g.

Cell Culture
The breast cancer cell lines MCF-7 and MDA-MB-231 were obtained from the American Type Culture Collection (ATCC). The cells were cultured in a humidified 95% air/5% CO 2 incubator at 37 • C in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, and 100 IU/mL penicillin.

Cytotoxicity Studies
MCF-7 and MDA-MB-231 cells were seeded on 12-well plates at a density of 5 × 10 4 and cultured to 70% confluency. The synthesized compounds were dissolved in sterile ddH 2 O, and appropriate serial dilutions were prepared for further use. Following an overnight starvation in serum-free medium, the cells were incubated with AuNRs@PEG and AuNRs@PEG@NAP in different concentrations (1, 50, and 200 µg/mL) for 24 h. The adherent cells were subsequently harvested and counted manually, and the cytotoxicity was estimated as a percentage of living cells relative to non-treated control cells ± standard deviation (SD) of experiments in triplicate. The graphs were created with GraphPad Prism 8.

Conclusions
A facile experimental protocol for the synthesis of (PEGylated) gold nanorods (AuNRs@PEG) with AR = 6.0 is presented as well as an effective drug-loading procedure using non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP). Drug release studies revealed a pH-sensitive behavior for AuNRs@PEG@NAP while the PEG content seems to determine the rate of release with a fast release up to a maximum of 24% and 33% NAP within the first 180 min at pH = 4.2 and 6.8, respectively.
The interaction of AuNRs with CT DNA was studied by UV-vis spectroscopy at three different temperatures and different interaction modes were concluded for each AuNR. More specifically, AuNRs@PEG may interact with CT DNA via van der Waals forces and hydrogen bonds on the external surface (groove-binding), while for AuNRs@PEG@NAP, π-π stacking interactions may be developed with CT DNA resulting in the possible intercalation of the naproxen moiety in-between DNA bases, which was further confirmed via its ability to displace the classical intercalator ethidium bromide from the EB-DNA adduct. The AuNRs may induce the moderate cleavage to pBR322 plasmid DNA at a rather high concentration (0.25 mg/mL) and AuNRs@PEG@NAP (48% of DNA cleavage) is more active than AuNRs@PEG.
The AuNRs studied herein showed a noteworthy affinity for bovine and human serum albumins and may become reversibly and tightly bound to them. In most studies, the affinity of the AuNR loaded with naproxen (AuNRs@PEG@NAP) for the albumins presents two to three times higher than that of AuNRs@PEG. The binding constants of both AuNRs studied herein to biomacromolecules are comparable with other reported similar systems including gold nanoparticles, gold nanoclusters, and carriers of naproxen (such as carbon nanotubes and spin-crossover nanoparticles), but slightly lower than most metal-naproxen complexes reported. Both AuNRs@PEG and AuNRs@PEG@NAP exerted similar cytotoxic effects against MCF-7 cells and MDA-MB-231 cancer cells and were more active against MDA-MB-231 cells. The exploration of loading/release mechanisms and DNA-binding studies for the case of other NSAID and anticancer drugs as well as the role of the molecular weight of mPEG-SH derivatives in the loading/release mechanism is under further study.